Steroidogenic factor-1 (SF-1) plays pivotal roles in endocrine function and gonad development. Indeed, human patients with mutations in SF-1 are sex-reversed, lack normal gonad structure, and suffer from severe adrenal insufficiency. The SF-1 knock out mouse also has agenesis of gonads and adrenal glands. Sex-specific roles of SF-1 in gonad differentiation remain unknown because gonads in SF-1 disrupted animals are lost before the onset of sex determination. The main goal of this proposal is to identify the sex-specific functions of SF-1 during mouse gonad development. To examine the role of SF-1 during gonad development in both sexes, we have developed a technique to introduce nucleic acids into the urogenital ridge and monitor the ensuing consequences in culture. Our overall hypothesis is that the sexually dimorphic expression pattern of SF-1 is integral for male versus female gonadogenesis and is caused by sex-specific regulation of the SF-1 promoter. First, we will determine the consequences of SF-1 misexpression by targeting siRNAs specific for SF-1 in explant cultures of male gonads (Aim 1). Second, we will evaluate the 5' regulatory region of SF-1 in male and female gonad explant cultures to ascertain whether there are specific elements that target sex-specific expression of the gene (Aim 2). We have effectively eliminated the presence of SF-1 in siRNA treated gonads and have successfully targeted SF-1 promoter constructs to the correct cell types in males and females. These studies are the first known attempt to investigate the mechanism of SF-1 action in a live, intact, functioning gonad as it is developing. Findings resulting from these studies will have a profound impact on our current understanding of SF-1 function during gonad development. Furthermore, this method will enable scientists to embark on new studies on genes, known and unknown, that will lead to significant advances in the field of gonadogenesis and provide hope for a cure of gonadal anomalies including birth defects, infertility, and cancers. [unreadable] [unreadable]